Process for the manufacture of isavuconazole or ravuconazole

ABSTRACT

The invention relates to a process for the manufacture of diastereomerically and enantiomerically enriched triazole compounds isavuconazole and ravuconazole, comprising a Reformatsky reaction between a ketone and a 2-halozincpropionate ester, followed by a resolution step, preferably an enzymatic resolution with an esterase enzyme.

The invention relates to a process for the manufacture of adiastereomerically and enantiomerically enriched ester intermediate forisavuconazole or ravuconazole.

Isavuconazole and ravuconazole are triazole antifungal compounds.Processes for the manufacture of isavuconazole and ravuconazole weredisclosed in patents WO99/45008, WO2007/062542 and WO03/002498 toBasilea. In WO2011/042827 a process for the manufacture ofenantiomerically pure antifungal azoles such as ravuconazole andisavuconazole is disclosed, wherein a classical resolution of a racemicmixture is performed by the addition of an enantiopure chiral acid, thencollection of the desired diastereomer followed by conversion of thesalt into the enantiomerically pure form of the desired compound bytreatment with a base or an ion-exchange resin. The disadvantages ofusing such classical resolution are that the chiral auxiliary needs tobe applied in near stoichiometric amounts, and that additional processsteps are required for recovery of these relatively high amounts ofchiral reagent as well as for converting the salt into the freeenantiopure product.

Therefore, it is the object of the present invention to provide animproved process for the manufacture of isavuconazole or ravuconazolewith high diastereomeric and enantiomeric excess (d.e. and e.e.respectively).

“Enantiomerically enriched” as defined herein is equivalent to the term“optically active” and means that one of the enantiomers of a compoundis present in excess compared to the other enantiomer. This excess willhereinafter be referred to as “enantiomeric excess” or e.e. (as forexample determined by chiral GC or HPLC analysis). The enantiomericexcess e.e. is equal to the difference between the amounts ofenantiomers divided by the sum of the amounts of the enantiomers, whichquotient can be expressed as a percentage after multiplication by 100.

“Diastereomerically enriched” means that one of the diastereomers of acompound is present in excess compared to the other diastereomer. Thisexcess will hereinafter be referred to as “diastereomeric excess” ord.e. Similarly, diastereomeric excess d.e. is equal to the differencebetween the amounts of diastereomers divided by the sum of the amountsof the diastereomers, which quotient can be expressed as a percentageafter multiplication by 100.

The invention now relates to a process for the manufacture ofdiastereomerically enriched compounds according to formula (I),

wherein R₁ and R₂ are each fluoride or hydrogen and when R₁ is fluoride,R₂ is hydrogen and when R₂ is fluoride, R₁ is hydrogen, wherein R is aC₁-C₁₂ alkyl, a C₅-C₁₂aryl or a C₆-C₁₁ aralkyl,which comprises the steps:

-   (i) preparation of a 2-halozinc propionate ester according to    formula (II)

-   -   wherein X is bromide, iodide or chloride,    -   in the presence of a solvent,    -   at a temperature below the boiling temperature of the solvent,

-   (ii) introduction of a ketone according to formula (III)

-   (iii) a Reformatsky reaction between the 2-halozincpropionate ester    according to formula (II) and the ketone according to formula (III)    in the presence of a solvent, removal of the excess of zinc,    -   resulting in a precipitate of the desired        (2R,3R)/(2S,3S)-diastereomers of the ester according to formula        (I),    -   wherein the sequence in which steps (i) and (ii) are performed        can be interchanged.

More specifically, the present invention relates to a process for themanufacture of a mixture of diastereomers of a3-hydroxy-2-methyl-4-[1,2,4]triazol-1-yl-3-phenyl-butyric acid esterderivative according to formula (I):

-   -   which is enriched in the corresponding (2R,3R)/(2S,3S) racemate,        and    -   wherein R₁ and R₂ are each fluoride or hydrogen and when R₁ is        fluoride, R₂ is hydrogen and when R₂ is fluoride, R₁ is        hydrogen, wherein R is a C₁-C₁₂ alkyl, a C₅-C₁₂ aryl or a C₆-C₁₁        aralkyl,    -   which comprises steps    -   (i) preparation of a 2-halozinc propionate ester according to        formula (II)

-   -   -   wherein X is bromide, iodide or chloride,        -   in the presence of a solvent,        -   at a temperature below the boiling temperature of the            solvent,

    -   (ii) introduction of a ketone according to formula (III)

-   -   (iii) performing a Reformatsky reaction between the        2-halozincpropionate ester according to formula (II) and the        ketone according to formula (III), in the presence of a solvent,        -   allowing the resulting reaction mixture to form a            precipitate by leaving the mixture stand, with or without            stiffing, for more than 0.5 hours preferably for more than 2            hours, after addition of the last reagent to the mixture,            wherein the precipitate is enriched in racemic            (2R,3R)/(2S,3S) ester according to formula (I), and        -   separating said precipitate,    -   wherein the sequence in which steps (i) and (ii) are performed        can be interchanged and wherein the excess of zinc is removed        before formation of said precipitation.

Surprisingly, this Reformatsky type reaction leads to diastereomericallyenriched isavuconazole and ravuconazole. In comparison with the methodsof the prior art, the process according to the invention requires simplereactants and conditions and delivers the desired isomer in high yield.

In EP0199474 the Reformatsky reaction was applied for the manufacture oftriazole compounds. It was disclosed that these compounds can beobtained in the form of racemic mixtures and that these mixtures can beseparated into the individual isomers by methods known in the art.However, successful enzymatic resolution of the racemic ester obtainedwith the Reformatsky reaction requires the ester to be scalable andcost-efficiently produced in a high diastereomeric purity. The estersobtained from Reformatsky reactions as disclosed in EP0199474 do notfulfil that requirement, as has been demonstrated in comparative exampleB of this application. Surprisingly, we have found that applying aReformatsky reaction wherein the Reformatsky reagent2-halozincpropionate ester is obtained at a temperature below theboiling temperature of the solvent and then allowing precipitationaccording to the present invention provides direct access to the desireddiastereomer of ester (I) in a very high d.e. (>97%) in a single step.

An alternative method for the preparation of the racemic ester (I) is acoupling reaction using an organic lithium salt. For example, WO9217474discloses a method for preparing ester (I) (R₂ is F) through a lithiumdiisopropylamide (LDA) mediated coupling of ethylpropionate to ketone(III) (R₂ is F) at −70° C. Column chromatography was applied to separatethe two diastereomers that are formed in the reaction (d.e. notreported), which is considered to be an inefficient and expensivepurification method on large scale. Similar results were obtainedin-house (see comparative example A): when ethylpropionate was coupledto ketone (III) (R₁ is F) in the presence of LDA at −78° C. the desiredester (I) (R₁ is F) was isolated in 61% yield with a poor diastereomericexcess (d.e.). of 29%. Hence, in view of

-   -   a) the poor diastereoselectivities and the concomitant low        yields of the reaction;    -   b) the absence of a cost-efficient and scalable method to        increase the d.e. after the reaction and    -   c) the use of anhydrous conditions at low temperature which is        associated with high costs        coupling reactions involving strong bases such as LDA (LiHMDS        etcetera) do not provide an industrially relevant entry into        esters of the general structure (I).

Diastereomeric excess measured after the Reformatsky reaction of theprocess according to the invention varies from 50 to 60% d.e. Afterprecipitation the product is isolated with diastereomeric excessesvarying between 97% and 99.9% d.e.

The product obtained after step (iii) of the process according to theinvention can be resolved to according to any known method, includinge.g. diastereomeric crystallization of the ester mixture aftersaponification of the ester and reaction of the obtained acid mixturewith an optically pure base like 1-phenylethylamine or2-amino-1-butanol, or chiral HPLC.

Subsequent enzymatic resolution of the (2R,3R)/(2S,3S)-ester (I) with anesterase enzyme is however preferred because it leads to a particularlyattractive industrially scalable route to isavuconazole or ravuconazolewith d.e.'s of more than 99% and e.e.'s of more than 99%. Such anenzymatic resolution approach has never been reported for (intermediatestowards) triazole-based anti-fungal agents despite the fact that thisclass of compounds has been in the centre of attention of thepharmaceutical industry for over 3 decades and despite the fact thatenzymatic resolution is a technology that is otherwise frequentlyemployed in pharmaceutical processes. Also a very recent patentapplication in the field (WO2011/042827), which has the resolution stepas the main subject of the invention, discloses classical resolutionsonly and not enzymatic resolution. Possibly, the relatively demandingsteric properties of triazole-based anti-fungal agents make themchallenging substrates for enzymes in general. It is clearly notstraightforward to find a suitable enzyme for this type of substrate. Infact over 200 hydrolytic enzymes were screened for the process accordingto the invention and only one type of enzyme family (i.e. esterases)provided both activity as well as selectivity towards esters of thegeneral formula (I).

In summary, the industrial preparation of the anti-fungal agentsisavuconazole and ravuconazole requires efficient and scalable methodsfor the introduction of both diastereo- as well as enantioselectivity.The herein reported diastereoselective Reformatsky-precipitationprotocol in conjunction with the enzymatic resolution procedure providesboth.

In a preferred embodiment of the present invention, formula (I)represents the ester intermediate for isavuconazole. When R₁ is fluorideand R₂ is hydrogen in formula (I), the ester intermediate forisavuconazole is represented. When R₁ is hydrogen and R₂ is fluoride informula (I), the ester intermediate for ravuconazole is represented.

R in the 2-halozincpropionate ester according to formula (II) can be abranched or unbranched C₁-C₁₂alkyl, a C₅-C₁₂ aryl or a C₆-C₁₁ aralkyl,preferably a branched or unbranched C₁-C₈ alkyl or C₅-C₈ aryl, morepreferably a branched or unbranched C₁-C₄ alkyl. A branched orunbranched C₁-C₄ alkyl can be any one from methyl, ethyl, n-propyl,i-propyl, n-butyl, sec-butyl, tert-butyl. An example for thearyl-2-halozincpropionate ester is phenol-2-halozincpropionate ester.Preferably, R is methyl or ethyl, more preferably R is ethyl.

X in the 2-halozincpropionate ester according to formula (II) can be anyone from bromide, iodide or chloride. More preferably X is bromide.

In an embodiment of the present invention, R in formula (II) is ethyland X in formula (II) is bromide.

The temperature applied in the Reformatsky reaction according to theinvention and more specifically in the manufacture of the2-halozincpropionate ester is at best low and may vary between −30° C.and the boiling temperature at atmospheric pressure of the solventapplied. At least the temperature is below the boiling temperature ofthe solvent at atmospheric pressure. At higher temperatures theformation of Reformatsky reagent is hampered, for example because ofhomocoupling of the esters with concomitant release of zinc salts thatinhibit the reaction, therewith preventing full conversion andinfluencing the precipitation. Preferably, the temperature is between−30° C. and 85° C., more preferably between −10° C. and 40° C. and mostpreferably between −10° C. and 10° C. Even more preferably, thetemperature is close to 0° C., e.g. between −2° C. and 2° C.

Accordingly, the temperature applied in step (i) of the processaccording to the invention may vary between −30° C. and the boilingtemperature at atmospheric pressure of the solvent applied. Preferably,the temperature in step (i) is below the boiling temperature of thesolvent at atmospheric pressure. More preferably, the temperature instep (i) is between −30° C. and 85° C., yet more preferably between −10°C. and 40° C. and most preferably between −10° C. and 10° C. Even morepreferably, the temperature in step (i) is close to 0° C., e.g. between−2° C. and 2° C.

Furthermore, the temperature applied in step (iii) of the processaccording to the invention may preferably vary between −30° C. and theboiling temperature at atmospheric pressure of the solvent applied. Morepreferably, the temperature in step (iii) is below the boilingtemperature of the solvent at atmospheric pressure. Even morepreferably, the temperature in step (iii) is between −30° C. and 85° C.,most preferably between −10° C. and 40° C. and even more preferablybetween 10° C. and 30° C. Still more preferably, the temperature in step(iii) is at room temperature, e.g. between 15° C. and 25° C.

The solvents applied in steps (i) and (iii) of the process of theinvention are aprotic solvents. Preferably, the solvents are polaraprotic solvents. To the alternative apolar aprotic solvents are used incombination with polar aprotic solvents. Suitable solvents aretetrahydrofuran, 2-methyl-tetrahydrofuran, tertbutylmethylether,di-isopropylether, di-ethylether, acetonitrile, ethylacetate,dichloromethane or toluene. Preferred solvents in steps (i) and (iii) ofthe process of the invention are independently tetrahydrofuran and2-methyl-tetrahydrofuran.

The solvents applied in steps (i) and (iii) of the process according tothe invention can be the same or different. More preferably, thesolvents applied in steps (i) and (iii) of the process according to thepresent invention are the same. Even more preferable the solvent insteps (i) and (iii) is tetrahydrofuran or 2-methyl-tetrahydrofuran.

The 2-halozincpropionate ester can be obtained via a reaction between a2-halopropionate ester and metallic zinc. Activation of zinc isdescribed by Fürstner (Chapter 14, The Reformatsky reaction inOrganozinc Reagents, Knochel and Jones, Oxford University Press, p287-305, 1999). The zinc applied in the process according to theinvention can advantageously be activated by acid or iodine treatment ofzinc or by reductive treatment of a zinc salt. Reductive treatment of azinc salt can be done with for example lithium, sodium, potassium ordiisobutylaluminiumhydride.

Furthermore, the particle size of the metallic zinc applied in theprocess according to the invention is preferably as small as possible.Smaller particles provide larger surface areas, thus enhancing theinteractions in the reaction. Preferably, the zinc particles have adiameter smaller than 50 μm, more preferably smaller than 10 μm, evenmore preferably smaller than 5 μm. Zinc particles of these sizes areoften referred to as zinc dust. In combination with the solvent, thezinc is often present as a suspension in the process according to theinvention. This suspension can be stirred during the Reformatskyreaction.

In the reaction between a 2-halopropionate ester and metallic zinc, thezinc is applied in 1 to 3 molar equivalents relative to the2-halopropionate, preferably in 1 to 2 molar equivalents, morepreferably in 1 to 1.2 molar equivalents relative to the2-halopropionate.

In the alternative, the 2-halozincpropionate ester according to formula(II) can be obtained via a reaction of the 2-halopropionate ester with adialkyl zinc reagent in the presence of a suitable metal catalyst. As anexample diethyl zinc and nickel (II) acetalacetonate as described byYang et al in Tetrahedron: Asymmetry (2007, 18, 949-962) can beemployed.

In step (iii) of the process according to the reaction, anhydrousconditions are preferred. Such conditions can be obtained by workingunder inert atmosphere, e.g. by applying nitrogen or argon. In an inertatmosphere according to the invention as little as possible water ispresent. The atmosphere is inert in that it is non-reactive in thechemical synthesis according to the invention.

In the process according to the invention, the sequence of preparationof the ester according to formula (II) (step (i)) and addition of theketone according to formula (III) (step (ii)) can be interchanged. Inone embodiment of the invention, the ketone was added after the2-halopropionate ester had reacted with the zinc to form the Reformatskyreagent (WO2009035684). In the alternative, the ketone is alreadypresent and the reactants for preparation of the 2-halozincpropionateester are added afterwards (Barbier conditions). The excess of zinc isremoved after completion of step (i) and before the precipitationstarts. The removal of excess of zinc can be done by filtering off.

After the Reformatsky reaction, the desired diastereomer of the esteraccording to formula (I) is allowed to precipitate. One of the factorsin allowing the ester to precipitate is leaving the reaction mixturestand for a certain period of time. Preferably the reaction is left formore than 12 hours after the addition of the last reagent, morepreferably for more than 6 hours, even more preferably for more than 2hours and most preferably for more than 0.5 hour. During the waitingtime, stirring of the reaction mixture can proceed as before.Precipitation can be enhanced by addition of a small amount ofprecipitate containing desired diastereomer, which was obtained before.Furthermore, precipitation can be stimulated and yield can be improvedby addition of a non-protic apolar solvent such as tertbutylmethyletheror n-heptane.

The precipitate obtained in step (iii) of the process according to theinvention is isolated through filtration. Subsequently the desireddiastereomer of the ester (I) is obtained by extraction into an organicsolvent such as ethyl acetate. Advantageously, this extraction involvestreatment with an aqueous acidic solution. Optionally, the organicsolution containing the ester (I) is concentrated to give a solid priorto the subsequent enzymatic resolution step.

Particularly preferred is a process according to the present invention,wherein the esterase enzyme used for resolution is an isolatedpolypeptide with esterase activity comprising an amino acid sequenceshown in SEQ ID No. 4 or a homologue thereof having an amino acididentity of at least 90%.

SEQ ID No. 4: MGQPASPPVVDTAQGRVLGKYVSLEGLAQPVAVFLGVPFAKPPLGSLRFAPPQPAEPWSFVKNTTSYPPMCCQEPIGGQMLSDLFTNRKERLIPEFSEDCLYLNIYTPADLTKRGRLPVMVWIHGGGLVVGGASTYDGLALAAHENVVVVAIQYRLGIWGFFSTGDEHSRGNWGHLDQVAALHWVQENIANFGGDPGSVTIFGESAGGESVSVLVLSPLAKNLFHRAISESGVAFTAG L VRKDMKAAAKQIAVLAGCKTTTSAVFVHCLRQKSEDELLDLTLKMKFFALDLHGDPRESHPFLTTVVDGVLLPKMPEEILAEKDFNTVPYIVGINKQEFGWLLPTMMGFPLSEGKLDQKTATSLLWKSYPIANIPEELTPVATDKYLGGTDDPVKKKDLFLDLMGDVVFGVPSVTVARQHRDAGAPTYMYEFQYRPSFSSDKKPKTVIGDHGDEIFSVFGFPLLKGDAPEEEVSLSKTVMKFWANFARSGNPNGEGLPHWPMYDQEEGYLQIGVNTQAAKRLKGEEVAFWNDLLSKEAAKKPPKIKHAEL

The esterase shown in SEQ ID Nr. 4 and homologues thereof are describedin WO2009/004039 and WO2010/122175.

Preferably, said esterase enzyme has at least 95% identity with SEQ IDNO 4, more preferably at least 97%, even more preferably at least 98%and most preferably more than 99% identity with SEQ ID No. 4.

Even more preferred is a process according to the present invention,wherein the esterase enzyme is an isolated polypeptide with esteraseactivity comprising an amino acid sequence shown in SEQ ID No. 2 or ahomologue thereof having an amino acid identity of at least 90%, whichhomologue contains valine as amino acid in position 239 of said sequenceor the position corresponding thereto.

SEQ ID No. 2: MGQPASPPVVDTAQGRVLGKYVSLEGLAQPVAVFLGVPFAKPPLGSLRFAPPQPAEPWSFVKNTTSYPPMCCQEPIGGQMLSDLFTNRKERLIPEFSEDCLYLNIYTPADLTKRGRLPVMVWIHGGGLVVGGASTYDGLALAAHENVVVVAIQYRLGIWGFFSTGDEHSRGNWGHLDQVAALHWVQENIANFGGDPGSVTIFGESAGGESVSVLVLSPLAKNLFHRAISESGVAFTAG V VRKDMKAAAKQIAVLAGCKTTTSAVFVHCLRQKSEDELLDLTLKMKFFALDLHGDPRESHPFLTTVVDGVLLPKMPEEILAEKDFNTVPYIVGINKQEFGWLLPTMMGFPLSEGKLDQKTATSLLWKSYPIANIPEELTPVATDKYLGGTDDPVKKKDLFLDLMGDVVFGVPSVTVARQHRDAGAPTYMYEFQYRPSFSSDKKPKTVIGDHGDEIFSVFGFPLLKGDAPEEEVSLSKTVMKFWANFARSGNPNGEGLPHWPMYDQEEGYLQIGVNTQAAKRLKGEEVAFWNDLLSKEAAKKPPKIKHAEL

The mutation of the esterase enzyme of SEQ ID No. 4 (APLE) by replacingleucine in position 239 of said sequence with valine is known from WO2010/122175.

As is known, the numbering of amino acids is dependent on the speciesthe protein originates from. The numbering can also change as a resultof deletions or insertions. It is known, however, to a skilled personhow to align sequences. Thus, for the purposes of this application, thephrase “or corresponding thereto” is used to describe amino acidpositions that except for the number are the same as the position 239 inSEQ ID No. 2.

Preferably, the esterase enzyme has at least 95% identity with SEQ ID NO2, more preferably at least 97%, even more preferably at least 98% andmost preferably more than 99% identity with SEQ ID NO 2.

Enzymes belonging to this category are mostly pig liver esterases orvariants thereof. Therefore, in an embodiment, the invention alsorelates to the process according to the invention wherein the enzymaticresolution in step (iv) is performed by pig liver esterases or variantsthereof, in particular by an esterase enzyme of SEQ ID NO 2 or 4, mostpreferably SEQ ID NO 2.

In the present application “an esterase having at least 90% sequenceidentity to the amino acid sequence of (a reference sequence)” meansthat such protein is a homologue of the respective reference sequencehaving an amino acid sequence, which is for at least 90% identical tothe amino acid sequence of the reference sequence as determined insequence alignments performed with sequence alignment tools such asBLASTP (http://blast.ncbi.nlm.nih.gov/Blast), ClustalW(http://www.ebi.ac.uk/Tools/clustalw2) or Align Plus 5 (Scientific &Educational Software, Cary, N.C., USA).

For the purposes of the present application, the term homologue is alsomeant to include nucleic acid sequences (polynucleotide sequences) whichdiffer from another nucleic acid sequence due to the degeneracy of thegenetic code and encode the same polypeptide sequence.

Sequence identity or similarity is herein defined as a relationshipbetween two or more polypeptide sequences or two or more nucleic acidsequences, as determined by comparing the sequences. Usually, sequenceidentities or similarities are compared over the whole length of thesequences, but may however also be compared only for a part of thesequences aligning with each other. In the art, “identity” or“similarity” also means the degree of sequence relatedness betweenpolypeptide sequences or nucleic acid sequences, as the case may be, asdetermined by the match between such sequences. Preferred methods todetermine identity or similarity are designed to give the largest matchbetween the sequences tested. In context of this invention a preferredcomputer program method to determine identity and similarity between twosequences includes BLASTP and BLASTN (Altschul, S. F. et al., J. Mol.Biol. 1990, 215, 403-410, publicly available from NCBI and other sources(BLAST Manual, Altschul, S. et al., NCBI NLM NIH, Bethesda, Md., USA).Preferred parameters for polypeptide sequence comparison using BLASTPare gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferredparameters for nucleic acid sequence comparison using BLASTN are gapopen 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

In the enzymatic resolution according to the invention several reactionparameters can be varied such as solvent, co-solvent, pH, temperature,and substrate concentration in order to optimize the reaction.

Generally the solvent can be a mixture of water with a water-misciblesolvent, for example with an alcohol such as methanol, ethanol,isopropanol or tert-butanol, or with dioxane, tetrahydrofuran, acetoneor dimethyl sulfoxide or a two-phase system of water and of awater-immiscible solvent, for example an aromatic compound such astoluene or xylene, an alkane such as n-hexane, n-heptane or cyclohexane,or an ether such as diisopropyl ether or methyl tert-butyl ether.

The nature of the co-solvent in the enzymatic resolution according tothe invention plays a crucial role, since for example no conversion wasobserved when 2-methyltetrahydrofuran was used. Preferably tert-butanol,tert-butylacetate, methylisobutylketone or toluene are used asco-solvent. More preferably, toluene is used as co-solvent for theenzymatic resolution.

The effect of pH on the enzymatic activity is not critical. The pH ofthe reaction solution is between 4 and 11, preferably between 6 and 9.However, more preferably the pH optimum for the enzymatic resolutionaccording to the invention lies in the range between pH 7.5 and 8.

The reaction temperature for the conversion of the invention is normallybetween 0 and 90° C., preferably between 10 and 60° C. The enzymaticresolution reaction according to the invention is faster at highertemperatures. However, the enzyme activity decreases over time at 37° C.Therefore, the temperature during the enzymatic resolution reaction ismore preferably between 28 and 37° C.

Substrate concentrations for the enzymatic resolution can vary from 0.1to 50 weight percentage, preferably from 1 to 25 weight percentage, morepreferably from 2 to 10 weight percentage. Most preferably, thesubstrate concentration is between 4 and 6 weight percentage.

The esterase according to this invention may be used in any form. Theesterase may be used for example in the form of a dispersion, a solutionor in immobilized form. Furthermore, the esterase may be used forexample as crude enzyme, as a commercially available enzyme, as anenzyme further purified from a commercially available preparation, as anenzyme obtained from its source by a combination of known purificationmethods, in whole (optionally permeabilized and/or immobilized) cellsthat naturally or through genetic modification possess the requiredesterase activity, or in a lysate of cells with such activity.

After the enzymatic resolution step, product isolation can take place byconventional methods such as extraction, crystallization, columnchromatography and/or distillation.

The ester obtained after step (iv) of the process according to theinvention can be converted to the corresponding amide through methodsknown in the art, e.g. through treatment with ammonia. Subsequently, theamide is further converted to isavuconazole or ravuconazole via knownmethods, e.g. as was disclosed in WO03/002498. The amide can bedehydrated into the corresponding cyanide and the cyanide can beconverted into the corresponding thioamide through e.g. reaction with asulfide salt such as ammonium sulfide and finally the thioamide can beconverted into isavuconazole or ravuconazole via reaction with anappropriately substituted 4-cyanoacetophenone reagent such as e.g.α-bromo-4-cyanoacetophenone.

The invention further relates to all possible combinations of differentembodiments and/or preferred features according to the process accordingto the invention as described herein.

The invention will be elucidated with reference to the followingexamples, without however being restricted by these:

EXAMPLES Diastereomeric Excess of Ester (I) was Determined by GC

GC: HP-5 column (30 m×0.32 mm×0.25 μm); Init. Temp.: 50° C., 0 min., 20°C./min to 150° C., 150° C. for 0 min.; 10° C./min to 190° C., 190° C.for 2 min.; 20° C./min to 300° C., 300° C. for 0 min.; Retention times:2.06 min.: ethylpropionate; 3.25 min.: ethyl-2-bromopropionate; 9.17min.: ketone II (R₁=F); 12.82 min.: RS/SR-ester I; 12.90 min.:RR/SS-ester I

¹H-NMR of RR/SS-ester I (CDCl₃, 300 MHz) 6=1.04 (d, J=7.2 Hz, 3H), 1.34(t, J=7.2 Hz, 3H), 3.30 (q, J=7.2 Hz, 1H), 4.25 (q, J=7.2 Hz, 2H), 4.60(d, J=14.1 Hz, 1H), 4.89 (d, J=14.4 Hz), 6.95 (m, 2H), 7.20 (m, 1H),7.75 (s, 1H), 8.11 (s, 1H) ppm.

¹H-NMR of RS/SR-ester I (CDCl₃, 300 MHz) 6=0.98 (t, J=7.2 Hz, 3H), 1.41(d, J=7.2 Hz, 3H), 3.37 (q, J=7.2 Hz, 1H), 3.95 (m, 2H), 4.61 (d, J=13.8Hz, 1H), 4.83 (d, J=14.1 Hz), 6.97 (m, 3H), 7.71 (s, 1H), 8.08 (s, 1H)ppm.

Comparative Example A Preparation of Racemic Ester (I) by OrganolithiumCoupling a) Preparation of a Stock-Solution of Lithium-Diisopropylamide(LDA) in Tetrahydrofuran (THF):

Diisopropylamine (716 mg, 7.1 mmol, 1.05 eq) was dissolved in anhydrousTHF (21.3 mL) and the resulting solution was cooled to −78° C. under anitrogen atmosphere. Subsequently, n-BuLi (2.7 M solution in n-heptane,2.5 mL, 6.7 mmol, 1.0 eq) was added in a drop wise fashion over 15minutes and the reaction mixture was stirred at −78° C. for anadditional 15 minutes. Then the solution was warmed to 0° C. and stirredfor 30 minutes after which the stock solution was cooled to −78° C.again.

b) Coupling Reaction:

The thus obtained LDA-solution (3.66 mL, 0.98 mmol, 1.1 eq) wastransferred to a Schlenk vessel and ethylpropionate (100 mg, 0.98 mmol,1.1 eq.) was added in a drop wise fashion at −78° C. under a nitrogenatmosphere. The resulting mixture was stirred at −78° C. for 30 minutesand then 1-(2,5-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone (200mg, 0.90 mmol, 1.0 eq.) in THF (3.66 mL) was added in a drop wisefashion over 15 minutes. The reaction mixture was stirred for 2 hours at−78° C. and then quenched with acetic acid and warmed to roomtemperature. The mixture was diluted with aqueous saturated NH₄Cl andethylacetate. The aqueous layer was extracted with ethylacetate (2×) andthe combined organic layers were washed with brine, dried (Na₂SO₄),filtered and concentrated in vacuo to give a yellow oil containing theracemic ester I with a diastereomeric excess of 29% in favour of thedesired RR/SS diastereomer. Further purification by columnchromatography (n-heptane/EtOAc/MeOH 60/40/5 v/v/v) provided the RR/SSdiastereomer (light yellow solid) as well as the RS/SR diastereomer(off-white solid) in a combined overall yield of 179 mg (0.55 mmol,61%).

Comparative Example B Reformatsky Reaction According to Steps (i), (ii)and (iii) at Elevated Temperature with Ketone Already Present (BarbierConditions)

A 2-neck flask with cooler was charged with zinc (1.1 g, 17 mmol, 3.8eq.) and heated in vacuo using a hotgun (3 nitogen-vacuum cycles).Subsequently, THF (60 mL) was added and then trimethylsilylchloride(0.15 mL). The resulting suspension was stirred under a nitrogenatmosphere at room temperature for 15 minutes, after which a solution ofketone III (R₁=F, 1.0 g, 4.5 mmol, 1.0 eq.) in THF (30 mL) was added.The reaction mixture was then heated to 66° C., after which the heatingsource was removed. Subsequently, a solution of ethyl-2-bromopropionate(0.87 mL, 1.2 g, 6.7 mmol, 1.5 eq.) in THF (20 mL) was added dropwiseover 10 minutes. The reaction mixture was then stirred at 66° C. for 1.5hours, after which it was cooled to room temperature. The reaction wasquenched by addition of a saturated aqueous ammoniumchloride solution(100 mL) and diluted with methyl-tertbutyl ether (MTBE, 100 mL). Thelayers were separated and the aquous layer was extracted with MTBE(2×100 mL). The combined organic layers were washed with brine (100 mL),dried (Na₂SO₄), filtered and concentrated in vacuo to give a yellow oil(1.4 g) containing racemic ester I. ¹H-NMR- and GC-analysis showed aconversion of ketone III (R₁=F) of 80% and a d.e. of ester I of 60% infavor of the desired RR/SS-diastereomer. The product was not purifiedfurther.

Example 1 Reformatsky Reaction According to Step (iii) withPre-Formation of Reformatsky Reagent at Low Temperature Followed byAddition to the Ketone a) Preparation of Stock Solution of ReformatskyReagent:

A 2-neck flask was charged with zinc (5.8 g, 89 mmol, 2.0 eq.) under anitrogen atmosphere and anhydrous THF (101 mL) and thentrimethylsilylchloride (TMSCl, 1.12 mL) were added. The resultingsuspension was stirred at room temperature for 30 minutes and thencooled to 0° C. Subsequently, ethyl-2-bromopropionate (5.8 mL, 8.1 g,44.7 mmol, 1.0 eq.) was dosed to the suspension in a drop wise fashionover 30 minutes. The reaction mixture was stirred for an additional 15minutes and then filtered under a nitrogen atmosphere into a Schlenkvessel to remove residual zinc.

Ketone III (R₁=F, 1.0 g, 4.5 mmol, 1.0 eq.) was charged into a Schlenkvessel and anhydrous THF (10 mL) was added under a nitrogen atmosphere.To the resulting solution was added 20 mL of the previously preparedstock solution of Reformatsky reagent (vide supra, 8.36 mmol, 1.9 eq.)in a dropwise fashion over 30 minutes at room temperature whilestirring. After completion of the addition the resulting reactionmixture was stirred under a nitrogen atmosphere for 36 hours (clearsolution). GC-analysis showed that the ester I (R₁=F) had formed with80% conversion based on ketone III (R₁=F) and a d.e. of 60% in favor ofthe desired RR/SS diastereomer. The reaction mixture was concentrated invacuo to a volume of 10 mL after which n-heptane was added untilformation of a solid was observed. The resulting suspension was stirredfor 16 hours after which the solid was isolated through filtration. Thesolid was then dissolved in a mixture of aqueous HCl (pH=1) and ethylacetate resulting in a clear biphasic system. The phases were separatedand the aqueous layer was extracted with ethyl acetate (2×). Thecombined organic layers were washed with water and brine, dried(Na₂SO₄), filtered and concentrated in vacuo to give racemic RR/SS esterI (R₁=F) as a light yellow solid with >99% d.e. as determined by GC.

Example 2 Reformatsky Reaction According to Step (iii) with Zinc RemovalPrior to Addition of the Ketone

Zinc (11.7 g, 179 mmol, 4.0 eq.) was suspended in THF (200 mL) andstirred in the presence of TMSCl (2.25 mL) under a nitrogen atmosphereat ambient temperature for 30 minutes in a 250 mL 3-neck flask.Subsequently, the suspension was cooled to 0° C. andethyl-2-bromopropionate (11.6 mL, 89.6 mmol, 2.0 eq) was added via asyringe pump over 45 minutes. The reaction mixture was stirred for anadditional 15 minutes at 0° C. (conversion checked with GC to be 100%),after which the suspension was filtered via cannula over a glass filterunder a nitrogen stream to the reaction vessel (500 mL 3-neck flask).Subsequently, a solution of ketone III (R₁=F, 10 g, 44.8 mmol, 1.0 eq.)in THF (130 mL) was dosed to the reaction mixture over 1 hour at roomtemperature. The mixture was stirred for an additional 72 hours at whichpoint a solid had formed. The suspension was filtered and the off-whitesolid was suspended in EtOAc and dissolved by addition of water andaqueous HCl until a clear biphasic system was obtained (pH 1). Thelayers were separated and the aqueous layer was extracted with EtOAc(2×). The combined organic layers were washed with water and brine,dried (Na₂SO₄), filtered and concentrated in vacuo to give racemic RR/SSester I (R₁=F, 8.8 g, 27 mmol, 60%) as a light yellow solid with >99%d.e. as determined by GC. The filtrate was subjected to the same aqueouswork-up. GC-analysis showed that the remaining ketone was present in thefiltrate as well as racemic ester I with a d.e. of −25% (in favor of theundesired RS/SR diastereomer).

Example 3 Reformatsky Reaction According to Step (iii) with Zinc Removalafter Addition of the Ketone but Prior to the Start of Precipitation

Zinc (98 g, 1.5 mol, 4.0 eq.) was suspended in THF (1.7 L) andmechanically stirred in the presence of TMSCl (18.7 mL) under a nitrogenatmosphere at ambient temperature for 30 minutes. Subsequently, thesuspension was cooled to 0° C. and ethyl-2-bromopropionate (96.6 mL, 744mmol, 2.0 eq) was added via a syringe pump over 1 hour. The reactionmixture was stirred for 15 minutes at 0° C. (conversion checked with GCto be 100%), after which a solution of ketone III (R₁=F, 83 g, 372 mmol,1.0 eq.) in THF (830 mL) was dosed over 20 minutes at room temperature.The mixture was stirred for an additional 15 minutes (conversion checkedwith GC to be >90%) and then filtered over celite. The d.e. of thereaction mixture was determined to be 60% by GC. Upon stirring of thereaction mixture, a suspension started to form after 5 hours. Thesuspension was stirred for 88 hours at which point the d.e. of themother liquid had decreased to −10% (in favor of the undesired RS/SRdiastereomer). The suspension was filtered and the off-white solid waswashed with MTBE (2×125 mL). The solid was subsequently suspended inEtOAc (2.1 L) and dissolved by addition of water (1.25 L) and aqueousHCl (10% w/w; 76 g) until a clear biphasic system was obtained (pH 1.3).The layers were separated and the organic layer was washed with aqueousHCl (1.1 L, pH 1.1), aqueous NaHCO₃ (500 mL containing 0.60 g NaHCO₃),water (2×250 mL) and brine (250 mL). The organic layer was then dried(Na₂SO₄), filtered and concentrated in vacuo to give racemic RR/SS-esterI (54 g, 167 mmol, 45%) in 97% d.e.

Example 4 Enzymatic Resolution According to Step (iv)

To a potassium phosphate buffer solution (500 mL, 50 mM, pH 7.8) wasadded a suspension (100 mL) containing the esterase of SEQ ID NO 1 (10g, whole Escherichia coli cells expressing the recombinant esterase geneof SEQ ID NO 1 encoding the esterase of SEQ ID NO 2, prepared asdescribed in WO2010/122175). The pH was adapted to 7.8 and subsequentlya solution of racemic RR/SS ester I (R₁=F, 40 g, 123 mmol, 97% d.e.) intoluene (400 mL) was added. The resulting mixture was stirred at 28° C.while maintaining the pH at 7.8 via titration with NaOH (1M, aq.).Analysis by HPLC showed that the e.e. of the R,R-ester I was 98.5% after22 hours. The reaction was worked-up as described below after 26 hours.N.B. the reaction with S/C-ratio of 2:1 and 3:1 were both finishedwithin 20 hours; e.e of R,R-ester I>99%.

SEQ ID NO 1: ATGGGACAACCAGCTTCGCCGCCTGTCGTTGATACCGCTCAAGGACGAGTCTTGGGTAAGTACGTCTCTTTAGAGGGATTGGCACAACCGGTTGCTGTCTTCTTGGGAGTCCCTTTTGCTAAGCCACCTCTTGGATCTTTGAGGTTTGCCCCGCCGCAACCAGCAGAGCCATGGTCTTTCGTTAAGAACACTACTTCCTACCCTCCAATGTGTTGTCAAGAACCAATCGGAGGACAAATGCTTTCAGACCTATTCACTAACAGAAAGGAAAGGCTTATCCCGGAGTTCTCTGAGGATTGCCTTTACCTAAATATTTACACTCCTGCCGATTTGACAAAGAGGGGTAGGTTGCCGGTTATGGTTTGGATTCATGGAGGAGGTTTGGTTGTTGGCGGAGCATCCACTTATGACGGATTGGCTCTTGCCGCGCACGAGAACGTTGTTGTTGTTGCTATTCAATACCGTTTGGGTATTTGGGGATTTTTCTCCACAGGAGATGAGCATTCCCGTGGAAACTGGGGCCATTTAGATCAAGTTGCTGCATTGCATTGGGTCCAAGAAAACATTGCTAACTTCGGAGGTGATCCAGGTTCTGTTACTATTTTCGGAGAATCAGCAGGCGGAGAGAGTGTCTCTGTATTGGTTTTATCACCATTAGCTAAGAACCTTTTTCATCGTGCTATTTCCGAAAGTGGTGTTGCTTTTACCGCCGGTGTGGTCAGGAAGGATATGAAGGCCGCAGCCAAGCAGATCGCTGTCCTTGCAGGATGCAAAACTACTACTTCGGCAGTCTTCGTGCATTGTTTGCGTCAAAAGTCGGAAGATGAACTTTTAGACCTCACGTTGAAGATGAAATTCTTTGCCCTTGACTTACACGGAGATCCAAGGGAATCTCACCCTTTTTTGACCACTGTTGTTGACGGAGTTTTGTTGCCTAAGATGCCTGAGGAAATCTTGGCCGAGAAGGACTTTAACACCGTCCCATACATTGTTGGAATTAACAAGCAGGAGTTCGGATGGCTTTTGCCAACGATGATGGGATTTCCTCTTTCCGAGGGAAAGTTGGATCAAAAGACGGCTACGTCACTTTTGTGGAAGTCCTACCCAATTGCCAACATTCCTGAAGAGTTGACCCCAGTTGCTACCGATAAGTATTTAGGAGGAACAGATGATCCTGTCAAAAAGAAAGATTTGTTTTTGGATCTGATGGGAGACGTTGTTTTCGGCGTCCCATCAGTTACGGTTGCTCGTCAGCATAGGGACGCAGGAGCTCCAACTTACATGTATGAGTTCCAATATCGTCCATCTTTTTCATCGGATAAGAAACCTAAGACGGTTATTGGAGATCATGGAGACGAAATTTTTTCCGTCTTCGGCTTCCCATTGCTCAAAGGTGACGCTCCAGAGGAAGAAGTCAGTCTTTCTAAGACGGTTATGAAATTTTGGGCTAACTTCGCCCGTAGTGGAAACCCTAATGGAGAAGGATTGCCTCACTGGCCGATGTACGATCAAGAGGAGGGATACCTTCAAATTGGTGTCAACACTCAAGCAGCTAAGAGGTTGAAAGGCGAGGAGGTTGCTTTTTGGAACGACCTGTTGTCCAAGGAAGCAGCAAAGAAGCCACCTAAGATAAAGCACGCCGAATTGTAA

Work-Up:

Dicalite 4208 (20 g) was added to the reaction mixture and the resultingsuspension was stirred for 5 minutes. Subsequently, the mixture wasfiltered over a precoated (dicalite 4108) glass filter. The filter cakewas washed with toluene (2×200 mL) and the combined filtrate wasseparated. At this stage, the toluene layer was slightly emulsified so asecond filtration over a precoated filter was performed. The resultingbiphasic filtrate was separated and the aqueous layer was added to theearlier obtained aqueous phase. The combined aqueous layers were thenextracted with toluene (250 mL) giving a completely emulsified organicphase. The toluene layer was filtered over a precoated filter twice,upon which a clear biphasic system was obtained. The layers wereseparated and the combined organic layers were washed with aqueousNaHCO₃ (100 mL, 5 wt %). Finally, the organic layer was concentrated invacuo to give R,R-ester I as an off-white solid:

Using the thus obtained protocol, 210 g of racemic RR/SS-ester I (d.e.97%) was converted in five batches each containing 40-45 grams ofstarting material. The enantiopure ester R,R-ester I (d.e. 95%;e.e. >99.5%) was isolated in 48% yield (101 g, 311 mmol).

Analysis:

Determination of the e.e. of ester I was done by chiral HPLC. A singlemethod was developed separating the enantiomers of racemic RR/SS-ester Ias well as the enantiomers of the corresponding carboxylic acid: ColumnDaicel AD, 2×50×4.6 mm ID, particle size: 10 μm, eluent:heptane/MeOH/EtOH 95:2.1:2.9 v/v/v+0.05% trifluoroacetic acid+0.05%diethylamine; runtime: 15 min, Pressure: 10 bars, Flow: 1.8 mL/min,Temperature: 20° C., UV detection at 210 nm. Retention times:SS-enantiomer ester I: 2.15 min.; SS-enantiomer carboxylic acid: 3.02min; RR-enantiomer carboxylic acid: 4.31 min.; RR-enantiomer ester I:8.21 min.

The conversion was confirmed by measuring the concentration of both theester I as well as the carboxylic acid by HPLC:

Column Hypersil BDS-3, 250×4.6 mm ID, particle size, 5 μm, eluent A:0.15% formic acid and 0.025% triethylamine in Milli-Q; eluent B: 0.15%formic acid and 0.025% triethylamine in acetonitrile, gradient A:B=95:5(v/v) to 5:95 over 10 min, maintain at 5:95 for 5 min, to 95:5 over 3min, maintain at 95:5 for 5 min (t=23 min). Flow: 1.0 mL/min,temperature: 40° C., UV detection at 210 nm. Retention times: carboxylicacid: 9.55 min.; ester I 12.35 min.

Example 5 Enzyme Screening

In a screening of more than 200 hydrolase enzymes (lipases, esterases,proteases) for the hydrolysis of ester I 225 μl of each individualenzyme in 100 mM potassium phosphate buffer pH 7.5 was incubated with 2mg of ester I dissolved in tert-butanol in a final volume of 250 μl incapped glass vials and incubated at 28° C. on an IKA KS 130 shaker (IKA,Staufen, Germany) at 400 rpm. After overnight incubation 40 μl 0.5 Mphosphoric acid were added to each vial, subsequently diluted with 710μl methyl-tert-butylether (MTBE) and centrifuged for 20 min at 3500 rpmin an Avanti J-20XPI centrifuge equipped with a JS-5.3 rotor (BeckmanCoulter, Woerden, The Netherlands).

The enantiomeric excess (e.e.) of both the remaining ester as well asthe resulting carboxylic acid was determined by HPLC (as describedabove). The conversion was calculated by comparison of these two e.e.values:

conversion=[e.e. ester/(e.e. ester+e.e. carboxylic acid)]*100%

Out of this large hydrolase collection only 8 recombinant pig liveresterases could hydrolyse preferentially the undesired enantiomer ofester I (Table 1).

TABLE 1 results of enzyme screening Esterase e.e. ester I e.e. acidconversion [SEQ ID No.] % % % 2 92.1 94 50 4 69.6 94 42 6 18.6 97 16 810.3 99 9 10 78.4 74 51 12 15.1 64 19

This example shows that several recombinant pig liver esteraseshydrolyse ester I enantioselectively. Esterase enzymes showing the SEQID No.s 4, 6, 8, 10 or 12 can be prepared using Escherichia coli cellsexpressing the recombinant esterase genes of SEQ ID No.s 3, 5, 7, 9 or11, respectively encoding said esterases according to the description inWO2009/004093 and WO2010/122175.

Example 6 Retest of Recombinant Pig Liver Esterases

Based on the results of the initial enzyme screening, 5 enzymes wereselected for a retest at 250 mg scale. The selection of enzymes wasbased on activity and selectivity towards ester I. For each individualreaction 250 mg of ester I was dissolved in 1 ml tert-butanol.Subsequently 5 ml 100 mM potassium phosphate buffer pH 7.5 and 4 mlcell-free extract containing the respective overexpressed recombinantpig liver esterases were added in Metrohm 718 STAT Titrinos (Metrohm,Schiedam, The Netherlands) at enzyme/substrate ratios of 1 mg totalprotein per 1 mg ester I. The pH was kept constant at 7.5 with 1 M NaOH.At regular time points samples were analysed for the enantiomeric excess(e.e.) of both the remaining ester as well as the resulting carboxylicacid was determined by HPLC (as described above). The conversion wascalculated by comparison of these two e.e. values. The results are givenin Table 2.

TABLE 2 Conversion and e.e.s of pig liver esterases catalysed hydrolysisreaction of ester I to the corresponding carboxylic acid. SEQ ID NO. 2SEQ ID NO. 4 Time e.e. ester e.e. acid conversion e.e. ester e.e. acidconversion (h) I (%) (%) (%) I (%) (%) (%) 1 — — — 2.6 80.3 3.1 2 32.599.9 24.5 3.9 90.0 4.1 3 50.7 99.7 33.7 5.7 91.8 5.9 5 99.5 99.6 50.08.3 92.0 8.3 7 99.3 99.9 49.9 14.1 91.6 13.3 23 99.9 99.9 50.0 27.6 92.423.0 SEQ ID NO. 6 SEQ ID NO. 8 Time e.e. ester e.e. acid conversion e.e.ester e.e. acid conversion (h) I (%) (%) (%) I (%) (%) (%) 1 −0.5 99.90.5 1.2 99.9 1.2 3 1.2 99.9 1.2 1.5 83.2 1.8 5 2.5 96.9 2.5 1.0 99.9 1.07 3.3 96.6 3.3 2.3 99.9 2.3 23 12.7 92.9 12.0 5.9 99.3 5.6 SEQ ID NO. 10Time e.e. ester e.e. acid conversion (h) I (%) (%) (%) 1 5.7 98.8 5.4 29.0 98.2 8.4 5 13.5 99.9 11.9 7 17.2 94.8 15.4 — = not determined

The enantioselectivities (E) of the individual esterase reaction werecalculated from the conversion and the e.e. of the produced carboxylicacid according to the formula:

E=ln((1−(conversion/100)*(1+(e.e._(acid)/100))))/ln((1−(conversion/100)*(1−(e.e._(acid)/100))))

and given in Table 3.

TABLE 3 Enantioselectivity of the pig liver esterase catalysedhydrolysis of ester I Esterase Enantioselectivity [SEQ ID No.] E 2 >5004 30 6 30 8 >200 10 45

The recombinant pig liver esterase of SEQ ID NO. 2 was identified as thebest candidate with 50% conversion, an e.e of 99.5% for ester I after 5h and an excellent enantioselectivity of E>500.

Example 7 Influence of Solvents on Pig Liver Esterase Reactions

The influence of organic solvents on the hydrolysis of ester I by thepig liver esterase of SEQ ID NO. 2 was investigated using recombinant E.coli cells expressing the gene of SEQ ID NO. 1, which had been producedas described in WO2010/122175. To 0.5 g of ester I 7.5 ml of 50 mMpotassium phosphate buffer pH 7.8, 0.1 g of wet recombinant E. colicells containing the esterase of SEQ ID NO. 1 (in 1 ml 50 mM potassiumphosphate buffer pH 7.8) and 2.5 ml of organic solvent were added at 28°C. In separate reactions either toluene, methyl-isobutylketone,tert-butylacetate or 2-methyl-tetrahydrofurane were added as organicsolvent. As control 2.5 ml of 50 mM potassium phosphate buffer pH 7.8were added instead of an organic solvent.

The pH was kept constant at 7.8 with 1 M NaOH. At regular time pointssamples were analysed for the enantiomeric excess (e.e.) of both theremaining ester as well as the resulting carboxylic acid was determinedby HPLC (as described above). The conversion was calculated bycomparison of these two e.e. values (as described above). The resultsare given in table 4.

TABLE 4 Effect of organic solvents on the hydrolysis of R,R/S,S-Ester Iby the recombinant pig liver esterase of SEQ ID No. 2. time e.e acide.e. ester I conversion (h) (%) (%) (%) no solvent 2 4.6 94 4.7 3 6.898.3 6.5 4 7.6 98.2 7.2 7 12.2 97.1 11.2 22 40.5 98.4 29.2 toluene 210.1 98.0 9.3 3 15.8 98.0 13.9 4 21.7 98.0 18.1 7 37.1 98.0 27.5 22 99.399.2 50.0 tert-butyl-acetate 2 5.0 95.6 5.0 4 9.0 97.4 8.4 6 15.1 97.913.4 28.5 68.2 97.4 41.2 methyl-isobutylketone 2 5.0 85.0 5.2 7.5 5.395.0 5.6 24 16.4 95.0 14.7

The solvents tert-butyl-acetate and especially toluene had a clearpositive effect on the rate of ester I hydrolysis. With toluene ester Iis obtained at 50.0% conversion and 99.2% e.e. after 22 hours.

1. A process for the manufacture of a mixture of diastereomers of a 3-hydroxy-2-methyl-4-[1,2,4]triazol-1-yl-3-phenyl-butyric acid ester derivative according to formula (I):

which is enriched in the corresponding (2R,3R)/(2S,3S) racemate, and wherein R₁ and R₂ are each fluoride or hydrogen and when R₁ is fluoride, R₂ is hydrogen and when R₂ is fluoride, R₁ is hydrogen, wherein R is a C₁-C₁₂ alkyl, a C₅-C₁₂ aryl or a C₆-C₁₁ aralkyl, comprising the steps of: (i) preparation of a 2-halozinc propionate ester according to formula (II)

wherein X is bromide, iodide or chloride, in the presence of a solvent, at a temperature below the boiling temperature of the solvent, (ii) introduction of a ketone according to formula (III)

(iii) performing a Reformatsky reaction between the 2-halozincpropionate ester according to formula (II) and the ketone according to formula (III), in the presence of a solvent, allowing the resulting reaction mixture to form a precipitate by leaving the mixture stand, with or without stirring, for more than 0.5 hours preferably for more than 2 hours, after addition of the last reagent to the mixture, wherein the precipitate is enriched in racemic (2R,3R)/(2S,3S) ester according to formula (I), and separating said precipitate, wherein the sequence in which steps (i) and (ii) are performed can be interchanged and wherein the excess of zinc is removed before formation of said precipitation.
 2. The process according to claim 1, wherein R₁ in formula (I) is fluoride, and R₂ is hydrogen.
 3. The process according to claim 1, wherein R in formula (II) is ethyl and/or X in formula (II) is bromide.
 4. The process according to claim 1, wherein the temperature in step (i) is between −10° C. and 40° C.
 5. The process according to claim 4, wherein the temperature is between −10° C. and 10° C.
 6. The process according to claim 1, wherein the temperature in step (iii) is below the boiling temperature of the solvent.
 7. The process according to claim 1, wherein step (i) is performed before step (ii).
 8. The process according to claim 1, wherein the solvent in step (i) and/or step (iii) is a polar aprotic solvent.
 9. The process according to claim 8, wherein the solvent is tetrahydrofuran, 2-methyl-tetrahydrofuran, tertbutylmethylether, di-isopropylether, di-ethylether, acetonitrile, ethylacetate, dichloromethane or toluene.
 10. The process according to claim 1, wherein the 2-halozincpropionate ester of step (i) is obtained via reaction of a 2-halopropionate ester with metallic zinc.
 11. The process according to claim 1, which is followed (iv) by dissolving and/or extracting the precipitate obtained in step (iii) in an organic solvent and resolution of the (2R,3R)/(2S,3S) diastereomers in said solution to obtain a product enriched in the desired (2R,3R) enantiomer of the ester of formula (I):


12. The process according to claim 11, wherein an enzymatic resolution of the diastereomer of the ester according to formula (I) is performed using an esterase enzyme.
 13. The process according to claim 12, wherein said esterase enzyme is an isolated polypeptide with esterase activity comprising an amino acid sequence shown in SEQ ID No. 4 or a homologue thereof having an amino acid identity of at least 95%, preferably of at least 98%.
 14. The process according to claim 12, wherein said esterase enzyme is an isolated polypeptide with esterase activity comprising an amino acid sequence shown in SEQ ID No. 2 or a homologue thereof having an amino acid identity of at least 95%, which homologue contains valine as amino acid in position 239 or the position corresponding thereto.
 15. The process according to claim 14, wherein said homologues have an amino acid identity of at least 98%, preferably of at least 99%, and contain valine as amino acid in the position 239 of the amino acid sequence according to SEQ ID No. 2 or the position of the sequence of the homologue corresponding thereto.
 16. The process according to claim 14, wherein the isolated polypeptide with esterase activity comprises the amino acid sequence shown in SEQ ID No.
 2. 17. The process according to claim 16, wherein the isolated polypeptide with esterase activity is the amino acid sequence shown in SEQ ID No.
 2. 18. The process according to claim 14, wherein an organic co-solvent is used in the enzymatic resolution, selected from tert-butanol, tert-butylacetate, methylisobutylketone and toluene.
 19. The process according to claim 11, followed by conversion of the product enriched in the desired (2R,3R) enantiomer of the ester of formula (I) obtained in step (iv) into the corresponding amide through treatment with ammonia.
 20. The process according to claim 19, followed by dehydration of the amide into the corresponding cyanide.
 21. The process according to claim 20, followed by conversion of the cyanide into the corresponding thioamide and, optionally, further conversion of said thioamide into isavuconazole, when the phenyl moiety of said thioamide is a 2,5-difluoro-substituted, or ravuconazol, when the phenyl moiety of said thioamide is a 2,4-difluoro-substituted, via reaction with an alpha-keto-substituted 4-cyanoacetophenone reagent.
 22. A mixture of 3-Hydroxy-2-methyl-4-[1,2,4]triazol-1-yl-3-phenyl-butyric acid ester diastereomers of formula (I):

comprising the racemic mixture of (2R,3R)/(2S,3S) esters at a diastereomeric excess, as determined by GC, between 97% and 99.9%, preferably between 99% and 99.9%, wherein R₁ and R₂ are each fluoro or hydrogen and when R₁ is fluoro, R₂ is hydrogen and when R₂ is fluoro, R₁ is hydrogen, wherein R is a C₁-C₁₂alkyl, a C₅-C₁₂aryl or a C₆-C₁₁aralkyl.
 23. A (2R,3R)-3-Hydroxy-2-methyl-4-[1,2,4]triazol-1-yl-3-phenyl-butyric acid ester derivative according to formula (I):

wherein R is a C₁-C₁₂ alkyl or C₅-C₁₂aryl, R₁ is fluoro and R₂ is hydrogen. 